Application of mirabilis himalaica to preparation of product with whitening and antioxidant effects

Abstract

The invention relates to the field of natural medicinal chemistry, in particular to application of mirabilis himalaica to preparation of a product with whitening and anti-oxidation effects, and innovatively adopts mirabilis himalaica to research the whitening and anti-oxidation effects. The mirabilis himalaica-derived extracellular vesicle-like nanoparticles are found to be capable of reducing the content of melanin in melanoma cells at the cellular level and inhibiting the activity of tyrosinase. In addition, the mirabilis himalaica source extracellular vesicle-like nanoparticles can also scavenge free radical ions, inhibit the generation of active oxygen in cells and promote the improvement of cell viability. The mirabilis himalaica-derived extracellular vesicle-like nanoparticles disclosed by the invention can be sufficiently internalized and absorbed by melanoma cells, the melanin content in the melanoma cells is remarkably reduced, melanin pigmentation on the epidermis of a mouse is remarkably reduced, and the mirabilis himalaica-derived extracellular vesicle-like nanoparticles also have excellent oxidation resistance. The method has potential for preparing products with whitening and antioxidant effects.

Description

Technical Field

[0001] The present invention relates to the field of natural medicinal chemistry, and in particular to application of Mirabilis jalapa Himalayanus in preparing products with whitening and antioxidant effects. Background Art

[0002] As people prioritize health, their focus on skin health is also increasing, and the development of whitening pharmaceutical products has become a research priority. Modern whitening ingredients, such as hydroquinone, vitamin C, and its derivatives, are primarily synthetic. However, while these compounds offer therapeutic benefits, they often come with side effects, such as skin damage. Consequently, research is turning to natural herbal remedies for whitening, offering advantages such as minimal side effects and superior penetration.

[0003] Plant-derived extracellular vesicle-like nanoparticles (PELNs) are bilayer nanoparticles secreted by plant cells and contain active substances such as proteins, lipids, and nucleic acids. The lipid bilayer structure of PLNs gives them excellent biocompatibility and stability, and they have diverse applications such as mediating intercellular communication, drug delivery, and immune regulation, holding great potential for application in skin beauty.

[0004] Himalayan Mirabilis jalapa (Mirabilis himalaica), known as "Bazhu" in Tibetan, primarily grows at altitudes of approximately 2,800-3,400 meters on the Tibetan Plateau. It is a perennial herbaceous plant belonging to the genus Mirabilis in the family Nyctaginaceae and is an important medicinal plant in Tibetan medicine. Its seeds and rhizomes are rich in flavonoids, alkaloids, and polysaccharides, exhibiting antioxidant, anti-inflammatory, and skin regeneration properties. They have been used in skin-improving and anti-aging cosmetics. However, there is little prior research on the whitening and antioxidant properties of Himalayan Mirabilis jalapa. Summary of the Invention

[0005] In response to the above technical problems, the present invention provides a use of Himalayan Mirabilis jalapa in preparing products with whitening and antioxidant effects.

[0006] A first aspect of the present invention provides a use of Himalayan Mirabilis jalapa in preparing a product with whitening and antioxidant effects.

[0007] A first aspect of the present invention provides a use of extracellular vesicles derived from Mirabilis jalapa Himalaya in preparing a product with whitening and antioxidant effects.

[0008] Preferably, the whitening effect is to inhibit tyrosinase activity, and the anti-oxidation effect is to scavenge free radical ions and inhibit the production of active oxygen in cells.

[0009] Preferably, the active ingredient of the Himalayan Mirabilis jalapa is used at a concentration of 50-200 μg / mL.

[0010] Preferably, the Himalayan Mirabilis jalapa includes fresh Himalayan Mirabilis jalapa and / or dried Himalayan Mirabilis jalapa.

[0011] Preferably, the preparation method of the Himalayan Mirabilis jalapa-derived extracellular vesicles comprises the following steps: mixing the Himalayan Mirabilis jalapa with water and then squeezing the juice to obtain the juice of the Himalayan Mirabilis jalapa raw material, filtering the extract and performing a first centrifugal separation, collecting the centrifugal precipitate, resuspending the extract, ultrasonically treating the extract, and filtering the extract through a liposome extruder to obtain a filtrate, centrifuging the filtrate for a second time, collecting the precipitate, and resuspending the precipitate to obtain the Himalayan Mirabilis jalapa-derived extracellular vesicles. Preferably, the first centrifugation is differential centrifugation.

[0012] Preferably, the parameters of the differential separation are: centrifugal force of 1000g for 1 hour, centrifugal force of 5000g for 1 hour, centrifugal force of 18000g for 2 hours, and the centrifugal sediment with a centrifugal force of 18000g is taken.

[0013] Preferably, the Mirabilis jalapa Himalaya-derived extracellular vesicles are extracted from the roots of Mirabilis jalapa Himalaya.

[0014] A third aspect of the present invention provides a whitening and antioxidant product, the active ingredient of which is Himalayan Mirabilis jalapa extract.

[0015] A fourth aspect of the present invention provides a whitening and antioxidant product, the active ingredient of which is extracellular vesicles of Mirabilis jalapa Himalayana.

[0016] Preferably, the derived extracellular vesicles are extracted from the roots of Mirabilis jalapa Himalayanus.

[0017] Preferably, the whitening and antioxidant product is any one of a skin external preparation, a medicine and a health food.

[0018] Preferably, the skin external preparation is selected from: facial cream, lotion, gel, toner, essence, facial mask, eye cream, aerosol cleansing foam, spray, shower gel, gel or facial cleanser.

[0019] The beneficial effects of the present invention are: This study innovatively uses Himalayan Mirabilis jalapa to study its whitening and antioxidant properties. Extracellular vesicle-like nanoparticles derived from Himalayan Mirabilis jalapa have been shown to reduce melanin content in melanoma cells and inhibit tyrosinase activity at the cellular level (in vitro). Furthermore, these extracellular vesicle-like nanoparticles can scavenge free radical ions, inhibit the production of reactive oxygen species in cells, and promote cell vitality.

[0020] The Himalayan Mirabilis jalapa-derived extracellular vesicle-like nanoparticles of the present invention are fully internalized and absorbed by melanoma cells, significantly reducing the melanin content in melanoma cells and significantly reducing epidermal melanin pigmentation in mice. They also have excellent antioxidant capacity. They have the potential to be used in the preparation of products with whitening and antioxidant properties, providing a new strategy for skin whitening and anti-aging. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The present invention provides a preparation process for the extracellular vesicle-like nanoparticles derived from Mirabilis jalapa Himalaya. Figure 2 This is a transmission electron micrograph of the extracellular vesicle-like nanoparticles derived from Mirabilis jalapa himalayaensis of the present invention; Figure 3 This is a graph showing the particle size distribution of the extracellular vesicle-like nanoparticles derived from Mirabilis jalapa Himalaya of the present invention; Figure 4 This is a graph showing the cytotoxicity of the extracellular vesicle-like nanoparticles derived from Mirabilis jalapa to melanoma cells and epidermal cells; Figure 5 This is a graph showing the uptake of the extracellular vesicle-like nanoparticles derived from Mirabilis jalapa by melanoma cells; Figure 6 This is a graph showing the results of the Mirabilis jalapa Himalaya-derived extracellular vesicle-like nanoparticles of the present invention reducing melanin content in melanoma cells; Figure 7 This is a graph showing the results of the Mirabilis jalapa-derived extracellular vesicle-like nanoparticles of the present invention reducing the activity of melanin synthesis proteins in melanoma cells; Figure 8 This is a diagram showing the free radical scavenging ability of the extracellular vesicle-like nanoparticles derived from Mirabilis jalapa himalaya of the present invention; Figure 9 This is a diagram showing the ability of the Mirabilis jalapa Himalayanus-derived extracellular vesicle-like nanoparticles of the present invention to protect cells and tissues from producing reactive oxygen species; Figure 10 This figure shows the therapeutic effect of the extracellular vesicle-like nanoparticles derived from Mirabilis jalapa of the present invention on the UVB-induced melanin production model of C57BL / 6 mice; Figure 11 This is a histological analysis of the therapeutic effect of the Himalayan Mirabilis jalapa-derived extracellular vesicle-like nanoparticles of the present invention on the UVB-induced melanin production model of C57BL / 6 mice. DETAILED DESCRIPTION

[0022] The present invention is described in detail below in conjunction with the embodiments and drawings. The following embodiments are implemented on the premise of the technical solution of the present invention, and provide detailed implementation methods and specific operating processes, but the protection scope of the present invention is not limited to the following embodiments.

[0023] In the following examples, unless otherwise specified, all methods are conventional methods; the reagents and materials described, unless otherwise specified, can be obtained from commercial sources.

[0024] The present invention relates to the whitening and antioxidant effects of Mirabilis jalapa Himalayas, and discovers for the first time that extracellular vesicles derived from Mirabilis jalapa Himalayas can achieve the whitening and antioxidant effects.

[0025] Example 1 like Figure 1 As shown, a method for preparing extracellular vesicle-like nanoparticles derived from Mirabilis jalapa Himalayana comprises the following steps: 1) Weigh an appropriate amount of fresh Himalayan Mirabilis jalapa and add an equal amount of purified water in a 1:1 ratio. Squeeze the juice to obtain the Himalayan Mirabilis jalapa juice. Filter through medical gauze and discard the residue.

[0026] 2) The plant extract was subjected to differential centrifugation at 1,000 g for 1 h, 5,000 g for 1 h, and 18,000 g for 2 h, and the precipitate was collected after centrifugation at 18,000 g. 3) After resuspending in PBS, the cells were sonicated and filtered through a 200 nm filter membrane of a liposome extruder to obtain the filtrate.

[0027] 4) The filtrate is subjected to ultracentrifugation, the precipitate is collected, and the precipitate is resuspended to obtain Mirabilis jalapa Himalayanus-derived extracellular vesicle-like nanoparticles (MEVs).

[0028] Figure 2 The figure shows the transmission electron microscopy observation results of the Himalayan Mirabilis jalapa-derived extracellular vesicle-like nanoparticles extracted in this example. From the figure, it can be seen that the characteristics of the Himalayan Mirabilis jalapa-derived extracellular vesicle-like nanoparticles observed under an electron microscope are that their morphology is a double-layer membrane structure and the particle size is about 200 nm.

[0029] The particle size distribution and dispersion index of the vesicles were detected using a nanoparticle size analyzer. Figure 3 The figure shows the particle size distribution results of the Himalayan Mirabilis jalapa-derived extracellular vesicle-like nanoparticles extracted in this example. It can be seen from the figure that the average particle size is 179 nm, which is consistent with the exosome particle size range.

[0030] Example 2 Toxicity test of extracellular vesicle-like nanoparticles (MEVs) derived from Mirabilis jalapa on cells: 1) Dilute the cell suspension and plate into a 96-well plate. Determine the cell number based on experimental requirements and cell type (generally between 3,000 and 5,000). Incubate in a CO2 incubator for 24 hours.

[0031] 2) After sterilization, the collected MEVs were filtered through a 0.22 μm filter, diluted with culture medium, and added to a 96-well plate. Different concentrations of MEVs (0, 12.5, 25, 50, 100, and 200 μg / mL) were added to the cells and incubated for 24 and 48 hours.

[0032] 3) Prepare the CCK8 reaction solution at a ratio of culture medium to CCK8 solution of 10:1. Aspirate the culture medium from the 96-well plate, then add 100 μL of the CCK8 reaction solution to the wells to be tested and shake thoroughly. Continue incubating in a CO2 incubator for 1 hour.

[0033] 4) Read the absorbance at 450 nm using a microplate reader and process the data to determine whether MEVs are toxic to cells.

[0034] Concentration gradients of engineered vesicles were added to human skin keratinocytes (HaCaT) and mouse melanoma cells (B16F10), and then incubated for 24 and 48 hours, respectively, and then CCK8 was used to detect cell proliferation. Figure 4 The graph shows the cytotoxicity of Mirabilis jalapa-derived extracellular vesicle-like nanoparticles (EVs) against melanoma and epidermal cells. As can be seen, treatment of HaCaT and B16F10 cells with MEVs at varying concentrations for 24 and 48 hours showed no significant cytotoxicity. These results demonstrate that the vesicles are not significantly cytotoxic and do not harm cell proliferation.

[0035] Example 3 Whitening efficacy test of Himalayan Mirabilis jalapa-derived extracellular vesicles Uptake of Mirabilis jalapa-derived extracellular vesicle-like nanoparticles (MEVs) by melanoma cells 1) Count the number of B16F10 cells and plate them into 12-well plates (1×10 5 The cells were cultured in a CO2 cell culture incubator for 24 h to allow them to adhere completely.

[0036] 2) Incubate the fluorescent dye DiI with MEVs at 37°C for 20 minutes, and then separate the dye by ultracentrifugation to obtain MEVs labeled with the fluorescent dye DiI. 3) DiI-labeled MEVs (200 μg) were added to the cells and incubated for 12, 24, and 48 h, respectively.

[0037] 4) Aspirate the culture medium, wash once with PBS, add 4% paraformaldehyde, and incubate at room temperature for 15 minutes to fix the cells. 5) Discard the fixative and wash three times with PBS. Add 500 μL of 0.2% Triton X-100 and treat for 10 minutes. 6) Discard the permeabilization agent and wash three times with PBS. Add diluted DAPI solution to the dish and incubate at room temperature for 2-5 minutes to label the cell nuclei. After the reaction is complete, wash three times with PBS.

[0038] 7) Observe the distribution of MEVs in B16F10 cells under an inverted microscope and keep the original image.

[0039] Figure 5 The results show the uptake of extracellular vesicle-like nanoparticles derived from Mirabilis jalapa by melanoma cells. As can be seen, some MEVs have already entered the cells at 12 hours, and significant red fluorescence is observed at 24 hours, with further enhancement at 48 hours. This indicates that B16F10 cells exhibit good uptake of MEVs.

[0040] Melanin detection in B16F10 cell supernatant: B16F10 cells were grown at 2×10 5 Cells were seeded at a density of 100 cells / well in 6-well plates overnight and treated with drugs after adherence. Each plate contained a control group, a 2μmol / L α-melanocyte-stimulating hormone (α-MSH) modeling group, a post-modeling positive drug treatment group (50μmol / L arbutin), and a post-modeling co-treatment group with MEVs at varying concentrations (50, 100, and 200μg / mL). After drug addition, the plates were incubated in a CO2-controlled incubator (5% CO2, 37°C) for 48 hours. 150μL of culture supernatant was aspirated and added to a 96-well plate, and the absorbance at 475nm was measured using a microplate reader.

[0041] Melanin detection in B16F10 cells: B16F10 cells were cultured at 2×10 5Cells were seeded at a density of 100 cells / well in 6-well plates overnight and treated with drugs after adherence. Each plate contained a control group, a 2 μmol / L α-MSH modeling group, a post-modeling positive drug treatment group (50 μmol / L arbutin), and a post-modeling co-treatment group with MEVs at varying concentrations (50, 100, and 200 μg / mL). After drug addition, the plates were incubated in a CO2 incubator (5% CO2, 37°C) for 48 hours. At the end of treatment, the cells were digested and then centrifuged at 900 g for 3 minutes. The cell pellet was dissolved in 200 μL of 1N 10% DMSO in NaOH at 80°C for 2 hours. Once the pellet was completely dissolved, it was transferred to a 96-well plate and its absorbance was measured at 475 nm using a microplate reader.

[0042] Figure 6 The results show that extracellular vesicle-like nanoparticles derived from Mirabilis jalapa can reduce melanin content in melanoma cells. The figure shows: (A) Melanin content in the cell supernatant. Melanin content in the supernatant culture medium of treated B16F10 cells was measured using a microplate reader. (B) Melanin content in the cells. Melanin in B16F10 cells treated with MEVs was dissolved using a specific concentration of sodium hydroxide, and absorbance was measured at 475 nm using a microplate reader.

[0043] The results showed that the positive control group had a higher melanin content than the negative control group. After the addition of the positive drug arbutin, the melanin content in the supernatant decreased and the color became lighter. After the addition of MEVs treatment, the melanin content in the supernatant decreased and the color became lighter as the concentration increased. And at a concentration of 200 μg / mL, it showed a better anti-melanin effect than arbutin. As shown in the figure, the right picture shows the melanin content in the cells after cell disruption. After treatment with arbutin, the color of the melanin extract became lighter and the melanin content decreased. After MEVs treatment, the 100 and 200 μg / mL groups showed a better effect of reducing intracellular melanin, and the effect was similar to that of arbutin treatment. It can be concluded that MEVs have a better effect in reducing melanin production.

[0044] Himalayan Mirabilis jalapa-derived extracellular vesicle-like nanoparticles reduce the activity of melanin synthesis proteins in melanoma cells: 1) During the experimental preparation phase, prepare an SDS-PAGE separation gel of appropriate concentration and sufficient running buffer and transfer buffer. After the fully solidified separation gel is fixed to the electrophoresis apparatus, inject running buffer to submerge the electrodes and detect any leaks.

[0045] 2) After leak detection, remove the comb and, in sequence, slowly add 20–30 μg of protein sample and a protein marker (as a control) to the sample wells using a pipette. Electrophoresis parameters are set as follows: Initially, use a constant voltage of 90 V to compress the sample. After the sample front migrates to the interface between the stacking gel and the separating gel (approximately 30 minutes), increase the voltage to 120 V and continue electrophoresis until the bromophenol blue tracer reaches the bottom edge of the gel. 3) Before transfer, thoroughly wet the PVDF / nitrocellulose membrane, filter paper, and sponge pad in transfer buffer. Assemble the transfer cassette in the following order: anode side (black cassette), stack the sponge pad, three layers of filter paper, separation gel, transfer membrane, three layers of filter paper, sponge pad, and cathode side (white cassette). Use a glass rod to roll and remove air bubbles from each interface during assembly. Place the transfer cassette correctly into the transfer tank, with the black side facing the cathode. Fill the cassette with pre-chilled transfer buffer until it completely submerges the transfer apparatus. Perform wet transfer at a constant current of 300mA for 90 minutes, while maintaining a low temperature by placing an ice pack in the transfer tank. 4) After transfer, use tweezers to transfer the NC membrane to an incubation box. Add blocking solution prepared with 5% skim milk powder to cover the NC membrane. Set the shaker speed and place it on a shaker at room temperature for 1 hour to block.

[0046] 5) Discard the blocking solution, wash with TBST, and cut the NC membrane according to the experimental requirements. After cutting, place the strips in an incubation box, add the prepared primary antibody, label, and incubate overnight at 4°C in a shaker.

[0047] 6) Recover the primary antibody and wash twice with TBST (10 min each). Then, add the secondary antibody diluted in skim milk and incubate on a shaker at room temperature for 1 h.

[0048] 7) Discard the secondary antibody and wash twice with TBST (10 min each). Prepare ECL developer according to the instructions. Cover the NC membrane with the developer, set the exposure times, and develop the membrane on a developer. Save the original image.

[0049] Figure 7 Shown are the results of Mirabilis jalapa-derived extracellular vesicle-like nanoparticles (EVs) reducing the activity of melanin synthesis proteins in melanoma cells. The figures show: (A) Western blot analysis of TYR protein expression in melanocytes treated with MEVs (the right figure is a grayscale analysis). (B) Western blot analysis of MITF protein expression in melanocytes treated with MEVs (the right figure is a grayscale analysis).

[0050] Compared to the α-MSH-stimulated melanin production model group, TYR protein expression decreased significantly after MEV treatment, and all treatment concentrations showed a significant inhibitory effect. Furthermore, the results showed that MEVs significantly inhibited TYR protein levels compared to the arbutin-treated group. The right panel shows that, compared to the α-MSH-stimulated melanin production model group, MEVs inhibited MITF protein expression. This effect of MEVs on MITF expression increased with increasing concentration, and the effect of the 200 μg / mL treatment group was significantly superior to that of the arbutin-treated group. These results demonstrate that MEVs inhibit the production of proteins in the intracellular melanin synthesis pathway, ultimately leading to the suppression of melanin production.

[0051] Example 4 Antioxidant efficacy test of extracellular vesicles derived from Mirabilis jalapa Himalaya: DPPH free radical scavenging experiment Accurately weigh 2.5 mg of DPPH and dilute to a 50 mL brown volumetric flask with anhydrous ethanol (final concentration 0.1 mM). Store at 4°C for short-term storage and -80°C for long-term storage. Add an appropriate amount of 0.5% TrionX-100 to the MEV sample solution and sonicate on ice for 30 minutes to obtain the treated MEV sample solution. Dilute the MEVs to final concentrations of 100, 200, 300, 400, and 500 μg / mL. Set up a blank group (1 mL PBS + 1 mL DPPH working solution), a control group (1 mL test sample solution + 1 mL anhydrous ethanol), an experimental group (1 mL test sample solution + 1 mL DPPH working solution), and a positive control group (using a vitamin C solution with the same concentration gradient instead of the sample solution as a positive control). Mix all solutions thoroughly and incubate in a 37°C water bath in the dark for 30 minutes. After the reaction is completed, the absorbance value (A) of each group is measured at 517nm using a microplate reader, and the DPPH free radical of each group is calculated according to the formula. DPPH free radical scavenging rate calculation formula: DPPH free radical scavenging rate (%) = (1-(A 实验组 -A 对照组 ) / A 空白组 ) A 实验组 : Absorbance of the mixed solution of sample and DPPH A 对照组 : Absorbance of the sample and ethanol mixture (eliminating the interference of the sample's own absorption) A 空白组 : Initial absorbance of DPPH and ethanol mixture ABTS cation radical scavenging ability test Weigh 2.45 mM potassium persulfate and 7 mM ABTS and dissolve them in 50 mL potassium phosphate buffer (pH 7.4). Mix thoroughly and incubate at 4°C in the dark for 12-16 hours to prepare the ABTS radical cation solution. Before use, dilute the ABTS stock solution with distilled water to an absorbance of 0.7 ± 0.02 at 734 nm. Prepare sample solutions at final concentrations of 100, 200, 300, 400, and 500 μg / mL. Set up a blank group (1 mL PBS + 1 mL ABTS working solution), a control group (1 mL test sample solution + 1 mL distilled water), an experimental group (1 mL test sample solution + 1 mL ABTS working solution), and a positive control group (using a vitamin C solution with the same concentration gradient instead of the sample solution as a positive control). Mix all solutions thoroughly and incubate in a 37°C water bath in the dark for 6 minutes. After the reaction, the absorbance (A) of each group was measured at 734 nm using a microplate reader, and the ABTS cation radical scavenging rate of each group was calculated according to the formula. ABTS cation radical scavenging rate calculation formula: ABTS cation clearance rate (%) = (1- (A 实验组 :-A 对照组 ) / A 空白组 ) A 实验组 :Absorbance of the mixture of sample and ABTS A 对照组 : Absorbance of the mixture of sample and distilled water (eliminating the interference of sample's own absorption) A 空白组 : Initial absorbance of the mixture of ABTS and distilled water FRAP total antioxidant capacity assay According to the kit instructions, Fe ions were prepared with distilled water to a final concentration of 0.05, 0.025, 0.0125, 0.00625, 0.003125, and 0.00156 μmol / mL. 2+ The standard solution was mixed with 500 μL FRAP working solution, and the mixture was stirred for 10 min. The absorbance was measured at 593 nm and the Fe 2+ Standard curve of the relationship between concentration and absorbance. Add an appropriate amount of cooled T-AOC extract to the vesicles, ultrasonically disrupt the vesicles for 10 minutes, and obtain the vesicle sample solution. Dilute the vesicle sample solution to 100, 200, 300, 400, and 500 μg / mL sample solutions. Set up a blank group (120 μL PBS + 900 μL FRAP working solution) and an experimental group (900 μL FRAP working solution sample solution to be tested + 900 μL FRAP working solution) respectively. After thorough mixing, react at room temperature for 10 minutes, measure the absorbance at 593 nm, and use the standard curve to calculate the corresponding Fe2+ The total oxidation capacity is calculated according to the formula: Total oxidative capacity μmol / ml = x × V 总 ÷V 样 V 总 : total reaction volume; V 样 : Sample volume in the reaction; x: Fe corresponding to the absorbance value calculated based on the standard curve 2+ concentration.

[0052] Figure 8 The graph shows the free radical scavenging capacity of extracellular vesicle-like nanoparticles derived from Mirabilis jalapa. The graph shows: (A) DPPH free radical scavenging assay results. The vertical axis represents DPPH scavenging rate (%), and the horizontal axis represents treatment concentration (μg / mL). (B) ABTS cation free radical scavenging assay results. The vertical axis represents ABTS scavenging rate (%), and the horizontal axis represents treatment concentration (μg / mL). (C) FRAP total antioxidant capacity assay results. The vertical axis represents FRAP total antioxidant capacity (μmol / mL), and the horizontal axis represents treatment concentration (μg / mL).

[0053] The results showed that after incubation with DPPH solution at 100, 200, 300, 400, and 500 μg / mL, MEVs showed a strong free radical scavenging ability, which reached more than 95%, and the free radical scavenging ability was significantly higher than that of vitamin C (V c ). ABTS was then used to test the antioxidant capacity of MEVs. The ABTS⁺ cationic radical (blue-green, 734nm absorbance) was reduced to colorless ABTS, and the decrease in absorbance reflected the overall antioxidant capacity of the exosomes. As shown in Result B, after incubation of MEVs with ABTS solution at 100, 200, 300, 400, and 500 μg / mL, the antioxidant capacity showed concentration gradient dependence. With the increase in concentration, the radical scavenging rate of Himalayan Mirabilis jalapa extracellular vesicles for ABTS gradually increased from about 20% to about 95%. At a concentration of 300 μg / mL, the ability to scavenge free radicals reached a plateau, which was also the highest ability to scavenge free radicals in this reaction system. At this time, it was the same as the level of free radical scavenging by Vc. The experimental results show that MEVs have excellent antioxidant capacity. Finally, this study used the FRAP iron reduction ability experiment to detect the ability of exosomes to remove Fe 3+ Reduction to Fe 2+ The absorbance increase is directly related to the content of reducing substances ( Figure 8C) can directly measure the potential of antioxidant components in MEVs to neutralize oxidative stress by donating electrons. The results showed that as the MEV concentration increased from 100 to 500 μg / mL, the antioxidant capacity of MEVs increased from 2.4 to 11 μmol / mL, demonstrating their strong reducing capacity. In summary, MEVs possess strong free radical scavenging and antioxidant activities, demonstrating their significant antioxidant potential.

[0054] Establishment of a hydrogen peroxide-induced oxidative damage model in HaCaT cells and assessment of their viability HaCaT cells in the logarithmic growth phase were seeded in 96-well cell culture plates, with 5×10 cells per well. 3 Cells were allowed to adhere overnight before drug addition. A mixture of hydrogen peroxide and culture medium was prepared with culture medium at concentrations of 100, 200, 300, 400, 500, 600, 700, 800, 900, and 1000 μmol / L, and a blank control group was set up. After 24 hours of treatment, the culture medium was aspirated, and the cells were washed three times with 1× PBS. Culture medium containing CCK8 reagent was added and cultured in a CO2 cell incubator for another 1 hour. The absorbance was measured at a wavelength of 450 nm using a microplate reader, and cell viability and IC were calculated. 50 .

[0055] Protection of HaCaT cell oxidative damage model by MEVs at different concentrations HaCaT cells in the logarithmic growth phase were seeded in 96-well cell culture plates, with 5×10 cells per well. 3 Cells were allowed to adhere overnight before drug addition. Cells were treated for 24 hours in culture medium containing various concentrations of MEVs (0, 100, 200, 300, 400, and 500 μg / mL) and a final hydrogen peroxide concentration of 550 μmol / L. The medium was aspirated, and cells were washed three times with 1× PBS. Cells were then incubated in a CO2 incubator containing CCK8 reagent for an additional hour. Absorbance was measured at 450 nm using a microplate reader to calculate cell viability.

[0056] MEVs scavenged reactive oxygen species in a HaCaT cell oxidative damage model HaCaT cells in the logarithmic growth phase were seeded in 24-well cell culture plates, with 5×10 cells per well. 4Cells were allowed to adhere overnight before drug addition. Cells were treated with either 550 μmol / L hydrogen peroxide or 550 μmol / L hydrogen peroxide plus 500 μg / mL MEVs, respectively. A blank control group was also established. After 24 hours of treatment, the culture medium was aspirated, the cells were washed three times with 1× PBS, and an appropriate volume of diluted DCFH-DA working solution was added. The cells were incubated in a CO2 incubator for another 30 minutes. Afterwards, the cells were washed three times with 1× PBS and observed under a fluorescence microscope.

[0057] Figure 9 The graph shows the ability of extracellular vesicle-like nanoparticles derived from Mirabilis jalapa to protect cells and tissues from reactive oxygen species (ROS) production. The figure shows: (A) HaCaT cells were treated with a gradient of hydrogen peroxide and incubated for 24 hours. Cell proliferation was assessed using CCK8 assays. Based on the cell proliferation results, the IC50 of hydrogen peroxide treatment was calculated to be 550 μg / mL. (B) An oxidative stress model was established in cells using 550 μg / mL of hydrogen peroxide. After incubation for 24 hours with varying amounts of MEVs, cell proliferation was assessed using CCK8 assays. (C) HaCaT cells were left untreated, treated with hydrogen peroxide, or treated with hydrogen peroxide and MEVs for 24 hours. After incubation for 30 minutes, a reactive oxygen species (ROS) fluorescent probe was added and observed under an inverted microscope for 30 minutes. Scale bar: 100 μm. The right panel shows the statistical results of the overall ROS fluorescence intensity.

[0058] The results showed that the cell viability showed a downward trend with the increase of hydrogen peroxide treatment concentration, and its IC 50 The concentration was 550 μmol / mL, so this concentration was selected for the subsequent oxidative stress model construction. Based on this model, MEVs were administered at concentrations ranging from 100 to 500 μg / mL. (B) shows that with increasing MEVs concentration, cell viability increased from approximately 50% to approximately 80%, demonstrating significant recovery. Further analysis using a reactive oxygen species (ROS) fluorescent probe revealed a significant increase in green fluorescence intensity within the cells treated with H2O2, indicating substantial ROS accumulation. However, after MEVs intervention, the fluorescence signal intensity significantly decreased, returning to near-normal levels (C). These results demonstrate that MEVs can effectively inhibit the abnormal production of ROS in cells under oxidative stress and significantly enhance cell viability, confirming their biological function in protecting cells from oxidative damage.

[0059] Testing the therapeutic effect of Himalayan Mirabilis jalapa-derived extracellular vesicle-like nanoparticles on the UVB-induced melanin production model in C57BL / 6 mice: 1) Six-week-old C57 mice were selected. After one week of adaptive feeding, the hair on their backs was removed using a depilatory cream and a razor. The mice were then exposed to UVB light for 45 minutes daily (radiation dose of 200 mJ / cm2) for one week.

[0060] 2) Mice with black spots on their backs were randomly divided into a blank group and an experimental group, with at least 6 mice in each group. Every day, 200 μL of 1,000 μg / mL MEVs and an equal volume of 1× PBS were evenly applied to the hair removal area on the back of the mice using a cotton swab. After one hour of treatment, the mice were irradiated with UVB light for 45 minutes (radiation dose of 200 mJ / cm 2 ) for three weeks. The mice's backs should be depilated weekly during this period, and the treatment effects should be continuously photographed and recorded.

[0061] 3) After treatment, remove the skin from the back of the mouse and fix it with 4% paraformaldehyde solution for subsequent sectioning.

[0062] Figure 10 for Figure 10 The therapeutic effect of Himalayan Mirabilis jalapa extracellular vesicle-like nanoparticles on a UVB-induced C57BL / 6 mouse melanogenesis model is shown in the figure: (A) Flowchart for establishing the mouse melanogenesis model and treatment. After one week of adaptive feeding, C57BL / 6 mice were shaved and continuously irradiated with UVB light for one week to establish the melanogenesis model. The mice were then treated with PBS and MEVs, respectively, and continuously irradiated with UVB light, and the treatment progress was recorded. (B) Comparison of melanogenesis model mice before and after treatment with PBS and MEVs. The area and degree of melanin pigmentation in the control group remained unchanged during the intervention period. In contrast, the melanin area in the MEVs-treated group decreased, and pigmentation was significantly reduced, with the skin appearance returning to near-normal levels. These results demonstrate that MEVs can effectively reverse UVB-induced skin hyperpigmentation by targeting the melanin synthesis pathway, providing key in vivo experimental evidence for their potential use as a novel skin-whitening agent.

[0063] To further elucidate the repair mechanism of MEVs against UVB-induced skin damage, this study conducted a multidimensional pathological analysis of mouse skin tissue, including hematoxylin-eosin (H&E) staining to assess epidermal structure, Fontana-Masson (FM) staining to quantify melanin granule deposition, and Masson trichrome staining to detect dynamic changes in collagen.

[0064] Figure 11Histological analysis of the therapeutic effect of Mirabilis jalapa-derived extracellular vesicle-like nanoparticles on the UVB-induced melanogenesis model in C57BL / 6 mice. The figures show: (A) H&E-stained images of the skin of the two groups of mice. Scale bar, 200 μm; (B) FM-stained images of the skin of the two groups of mice. Scale bar, 200 μm; (C) Masson's staining images of the skin of the two groups of mice. Scale bar, 200 μm. n = 6.

[0065] Figure A shows that after MEVs treatment, the epidermal thickness of mice decreased, mitigating UVB-induced photodamage and providing a protective effect. Figure B shows that after MEVs treatment, the number of melanin granules in mouse skin decreased significantly, and the skin color became lighter, further confirming the anti-melanin potential of MEVs in treating hyperpigmentation. Figure C shows that after MEVs treatment, the collagen content in mouse skin increased significantly, with dense and orderly collagen bundles, in stark contrast to the loose, broken fibers in the PBS control group. This demonstrates that MEVs can remodel the skin matrix by promoting type I / III collagen synthesis and reversing the degradation of connective tissue caused by photoaging.

[0066] The above multi-omics pathological evidence shows that MEVs not only exert whitening effects by regulating the melanin metabolic pathway, but also synergistically promote extracellular matrix regeneration, achieving multi-dimensional photodamage repair from the epidermis to the dermis.

[0067] Finally, it should be noted that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or make equivalent substitutions for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. Application of Himalayan Mirabilis jalapa in the preparation of products with whitening and antioxidant effects.

2. Application of Himalayan Mirabilis jalapa-derived extracellular vesicles in the preparation of products with whitening and antioxidant effects.

3. The use according to claim 1 or 2, characterized in that: The whitening effect is to inhibit the activity of tyrosinase, and the anti-oxidation effect is to scavenge free radical ions and inhibit the generation of active oxygen in cells.

4. The use according to claim 1 or 2, characterized in that: The effective component of the Himalayan Mirabilis jalapa is used at a concentration of 50-200 μg / mL.

5. The use according to claim 1 or 2, characterized in that: The Himalayan Mirabilis jalapa includes fresh Himalayan Mirabilis jalapa and / or dried Himalayan Mirabilis jalapa.

6. The use according to claim 2, characterized in that: The preparation method of the Himalayan Mirabilis jalapa-derived extracellular vesicles comprises the following steps: mixing the Himalayan Mirabilis jalapa with water and then squeezing the juice to obtain the Himalayan Mirabilis jalapa raw material juice; filtering the extract and performing a first centrifugal separation; collecting the centrifugal precipitate; resuspending the extract, performing an ultrasonic treatment, and filtering the extract through a liposome extruder to obtain a filtrate; performing a second centrifugal separation on the filtrate, collecting the precipitate; and resuspending the precipitate to obtain the Himalayan Mirabilis jalapa-derived extracellular vesicles.

7. The use according to claim 6, characterized in that: The first centrifugation is differential centrifugation.

8. The use according to claim 7, characterized in that: The parameters of the differential separation are: centrifugal force of 1000g for 1 hour, centrifugal force of 5000g for 1 hour, centrifugal force of 18000g for 2 hours, and the centrifugal sediment with a centrifugal force of 18000g is obtained.

9. A whitening and antioxidant product, characterized in that: Its active ingredient is Himalayan Mirabilis jalapa extract.

10. A whitening and antioxidant product, characterized in that: Its active ingredient is extracellular vesicles from Mirabilis jalapa Himalaya.

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